CN104726484B - Applications of the low phosphorus stress on rice transcription factor OsPHR2 in wheat - Google Patents

Abstract

The invention provides applications of the low phosphorus stress on rice transcription factor OsPHR2 in wheat, by the way that low phosphorus stress on rice transcription factor OsPHR2 is imported into Common Wheat Varieties Zheng wheat 0856 using particle gun mediated method, the Transgenic plant of wheat that can stablize heredity and expression is obtained.It is completed especially by following steps：The structure of OsPHR2 gene linear expression vectors；By the linear carrier transformed wheat callus containing target gene；The PCR identifications of resistance regeneration plant.Low phosphorus stress on rice transcription factor OsPHR2 linear expression vector is imported into Common Wheat Varieties Zheng wheat 0856 using particle gun mediated method, the Transgenic plant of wheat of heredity and expression can be stablized by obtaining, and efficiently technology and materials for support are provided using transgenic wheat new varieties for breeding high-yield high-quality resource.

Description

Applications of the low phosphorus stress on rice transcription factor OsPHR2 in wheat

Technical field

The invention belongs to transgenic breeding technical field, and in particular to OsPHR2 is in wheat for low phosphorus stress on rice transcription factor In application.

Background technology

Phosphorus element is one of a great number of elements necessary to plant growth and development, is the life macromolecules such as nucleic acid, phosphatide and ATP Important composition composition, played an important role within the time of infertility of plant, national overall survey of soil result shows, China There is nearly half to save more than the 50% soil available phosphorus content of (area) its gross area in below 5mg/kg, soil available phosphorus content to exist Below 10mg/kg's accounts for 80%-90%, and it is one of key constraints of crop production that soil, which lacks phosphorus,.When phosphorus is deficient in environment Reactive volt-ampere hour, the change of the change of phytomorph developmentally mainly root dry mass, including increase rhizome ratio, lateral root number and root hair Number and length etc..Degree etc. (Chen Lei etc., 2011；Abel et al,2011；Lopez-Bucio et al,2002；Ma et al,2001).Change in terms of Physiology and biochemistry includes the induced expression of high affine Pi transporters, soil secretion organic acid, Inner sources phosphorus The induced expression of sour enzyme and RNase, and increase organic acid and proton release, so as to come increase the absorption of phosphorus and recycling energy Power.

MYB-CC family gene members in myb transcription factor are to participate in Plant To Nutrient composition starvation stress regulatory process Important gene.Wykoff etc. has cloned AtPHR1 genes from arabidopsis first by the method for map based cloning, and proves to be somebody's turn to do Gene belongs to MYB-CC family members, take part in phosphate starvation answer signal pathway.Zhejiang University Wu Ping teaches problem Group has cloned 2 arabidopsis AtPHR1 homologous gene OsPHR1 and OsPHR2 from rice, and Phylogenetic analysis shows, OsPHR1 and OsPHR2 and AtPHR1 belongs to MYB-CC family members, and two genes are all by adjusting Identification of Phosphorus Starvation Express and participate in phosphate starvation signal transduction, there is similar function.But only OsPHR2 overexpression strain can be in normal phosphorus In the environment of make a large amount of enriched phosphorus of rice, and some phosphorus transporter albumen also raise accordingly.Under the conditions of low-phosphorous, overexpression OsPHR2 genes can improve response of the root system of plant to low-phosphorous signal, and the change of plant root dry mass is more strong compared with WT strain Strong, the result is that root increases, root hair increases, and P assimilation and utilization rate significantly improves.OsPHR2 is rice phosphorus signals-modulating system Important factor, absorption of the plant to phosphorus is remarkably improved under the conditions of soil lacks phosphorus.

By OsPHR2 channel genes into Common Wheat Varieties Zheng wheat 0856, expression can be stablized and lose the present invention by having formulated out Pass, marker-free, and P acquisition utilization ratio is compared with the transgenic wheat material that acceptor Zheng wheat 0856 significantly improves, and is seed selection Abridged edition high-efficiency high-quality transgenic wheat kind provides technology and materials for support.

The content of the invention

Present invention aims at applications of the low phosphorus stress on rice transcription factor OsPHR2 in wheat is provided, by constructing Low phosphorus stress on rice transcription factor OsPHR2 linear expression vector, common wheat product are conducted into using particle gun mediated method In kind Zheng wheat 0856, expression and heredity can be stablized by having formulated out, and P acquisition utilization ratio significantly improve compared with acceptor Zheng wheat 0856 Transgenic wheat material, efficiently provide technology and material branch using transgenic wheat new varieties for breeding high-yield high-quality resource Support.

The present invention realizes especially by following technical scheme：

The invention provides applications of the low phosphorus stress on rice transcription factor OsPHR2 in wheat, by by the low-phosphorous side of body of rice Compel transcription factor OsPHR2 imported into using particle gun mediated method in Common Wheat Varieties Zheng wheat 0856, acquisition can stablize heredity with The Transgenic plant of wheat of expression.It is completed especially by following steps：

1) structure of OsPHR2 genes linear expression vector；

2) by the linear carrier transformed wheat callus containing target gene；

3) the PCR identifications of resistance regeneration plant.

Further

Step (1) is specially to contain the ring-type expression vector pWMB003- of OsPHR2 genes using HindIII digestions OsPHR2, obtain the linear carrier containing target gene, piece segment length 3kb, be comprise only corn Ubiquitin promoters, The linear minimum expression cassette of OsPHR2 genes and Nos terminators.

Step (2) is specially using the rataria of Zheng wheat 0856 as acceptor material, after MS inducing culture light cultures 4-5d, is put Processing 4-6h is permeated in hypertonic culture medium, target gene linear carrier will be contained and containing bar gene linear carriers, and utilize gene Rifle mediated method cotransformation WHEAT CALLUS, 16h is continued with hypertonic culture medium, goes to calli induction media, culture 2 Careless fourth phosphine resistance screening regeneration culture medium, the generation of step sizing 2 are gone to after week, the resistance seedling of screening goes to root media, take root, Transplanted after strong sprout, obtain resistance regeneration plant.

Step (3) be specially extract regeneration plant genomic DNA, according to OsPHR2 gene orders devise a pair it is special Property primer PHR2P1：5'-AATATGGAGGCTTTGAACC-3' and PHR2P2：5'-TAACACACCCTTGGGAGTA-3', amplification Program is：95 DEG C 2 minutes；95 DEG C 20 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 38 circulation；72 DEG C 10 minutes, purpose fragment length For 476bp；PCR reaction systems are：10-50ng/ul genomic templates；10 μM of each 0.5 μ l of PC1 and PC2；2.5μl 10× Reaction Bu ffer；4μl dNTP mix(2.5mM)；0.5 μ l Taq enzymes, add water to 25 μ l.

Described MS inducing cultures are：The sucrose of MS+2mg/L 2,4-D+0.4% agar+3%, pH6.2；

Described hypertonic culture medium is：MS+2mg/L 2,4-D+0.7% agar+sucrose of mannitol+3%, pH5.8；

Described careless fourth phosphine resistance screening regeneration culture medium is：1/2MS+0.5mg/L NAA+2.0mg/L KT+0.4% fine jades The sucrose of fat+3%+4%PPT, pH6.2；

Described root media is：1/2MS+0.2mg/L the sucrose of NAA+0.4% agar+3%, pH6.5.

Present invention also offers a kind of linear expression vector containing target gene OsPHR2, piece segment length 3kb, be containing only There is the linear minimum expression cassette of corn Ubiquitin promoters, OsPHR2 genes and Nos terminators.

Beneficial effects of the present invention are：For a long time, both at home and abroad by wheat relative category and the phosphorus efficiency base of other species Major Technology because importing common wheat is by distant hybridization method, but does not obtain breakthrough always.Its is main There is reproduction between Spherical scanning and other species and wheat to isolate for reason.Technical solution of the present invention can break species it Between reproduction isolation, realize exchange of the gene in different plant species, therefore turn into and solve crops important character genetic improvement The Major Technology of bottleneck problem.、

Brief description of the drawings

Fig. 1 is plant expression vector pWMB003-OsPHR2 structural representations；

Fig. 2 is to turn OsPHR2 DNA triticum plant genomic DNA amplification electrophoretograms；1：OsT5-1；2,3,4,5：Feminine gender is planted Strain；6：OsT5-7；7：OsT5-28；8：OsT5-167；9：OsT5-183；10：Acceptor Zheng wheat 0856；11：Positive control；M： Marker；

Fig. 3 is transgenic wheat strain target gene OsPHR2 Southern hybridization analysis；M：1Kb DNALadder； +：Positive control (plasmid)；1,2,3：Acceptor Zheng wheat 0856；4,5,6：Transgenic line OsT5-28；1,4-BamHI digestions；2, 5-KpnI digestions；3,6-SacI digestions；

Fig. 4 is the RT-PCR detections of transgenic wheat OsPHR2 genes；,2,3：Transgenic line OsT5-28；ck：Do not turn Change control (Zheng wheat 0856)；Upper figure is the amplified production of OsPHR2 genes, and figure below is the amplified production of actin genes；

Fig. 5 is transgenic wheat strain OsT5-28 boot leaf Western results of hybridization；1：Transgenic line OsT5-28；2： Positive control rice；WT：Unconverted control (Zheng wheat 0856)；

Fig. 6 is that transgenic wheat strain and the most long root long of control compare under different condition；

Fig. 7 is that transgenic wheat strain and the root fresh weight of control compare under different condition.

Embodiment

With reference to embodiment, the present invention is described further, as described below, is only the preferable implementation to the present invention Example, is not limited the present invention, any person skilled in the art is possibly also with the disclosure above Technology contents be changed to the equivalent embodiment changed on an equal basis.It is every of the invention without departing from the present invention program content, foundation Technical spirit any simple modification that following examples are made or equivalent variations, all fall within protection scope of the present invention.

Embodiment 1

Experiment is purchased from hundred Imtech, Prime with plasmid extraction kit, Ago-Gel DNA QIAquick Gel Extraction Kits Star Taq DNA polymerases, Escherichia coli (Escherichia coli) bacterial strain DH5 α competent cell reagent preparation boxes are TaKaRa Products, restriction enzyme are MBI Products, and ampicillin is Amersco Products, remaining reagent It is pure for import or domestic analysis.

1) structure of OsPHR2 genes linear expression vector

Respectively with BamHI and KpnI difference double digestion pWMB003 carriers and the expression vector containing OsPHR2 genes, agar Sugared detected through gel electrophoresis, gel extraction purpose band and expression vector fragment, 22 DEG C of connections overnight, convert bacillus coli DH 5 alpha sense By state cell, pWMB003-OsPHR2 (Fig. 1) is accredited as through PCR and double digestion.

Contain the circular expression vector pWMB003-OsPHR2 of OsPHR2 genes, electrophoresis, recovery using HindIII digestions 3kb or so purpose fragment, the fragment are to comprise only promoter (corn Ubiquitin promoters), target gene (OsPHR2 bases Cause) and terminator (Nos terminators) linear minimum expression cassette.

2) OsPHR2 channel genes Zheng wheat 0856 and PCR identifications

Using the rataria of wheat breed Zheng wheat 0856 as acceptor material.Take in the middle part of Post flowering 12-14d fringe, be of the same size Immature seed, with 70% alcohol surface sterilization 1min, after aseptic water washing 3 times with 0.1% HgCl₂Sterilize 10min, nothing Bacterium water rinses 3-4 times.Rataria (1mm or so) is stripped out under aseptic condition, in MS inducing cultures (MS+2mg/L 2,4-D+0.4% The sucrose of agar+3%, pH6.2) on after light culture 4-5d, be placed in hypertonic culture medium (MS+2mg/L 2,4-D+0.7% agar+sweet Reveal the sucrose of alcohol+3%, pH5.8) on infiltration processing 4-6h, will contain target gene linear carrier (Ubiquitin promoters+ OsPHR2 gene+Nos terminators) and containing bar gene linear carriers, utilize particle gun mediated method cotransformation wheat callus group Knit, continue with 16h on hypertonic culture medium after biolistic bombardment, go to calli induction media.Careless fourth is gone to after cultivating 2 weeks Phosphine resistance screening regeneration culture medium (sucrose+4%PPT of 1/2MS+0.5mg/L NAA+2.0mg/L KT+0.4% agar+3%, PH6.2), in the generation of step sizing 2, per 2 weeks generations, the resistance seedling by screening goes to root media (1/2MS+0.2mg/L NAA+ The sucrose of 0.4% agar+3%, pH6.5) in, to regrowth root system it is more healthy and stronger when, you can carry out hardening 1-2 days.Take root, after strong sprout Move into flowerpot, basin alms bowl can be transplanted into by finally washing away the culture based draff of root system carrying, obtain 146 plants of resistance regeneration plant.

The genomic DNA of all regeneration plants is extracted, a pair of specific primers are devised according to OsPHR2 gene orders PHR2P1：5'-AATATGGAGGCTTTGAACC-3' and PHR2P2：5'-TAACACACCCTTGGGAGTA-3', amplification program are 95 DEG C 2 minutes；95 DEG C 20 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 38 circulation；72 DEG C 10 minutes.Purpose fragment length is 476bp., enter performing PCR detection using the primer pair regeneration plant, to identify positive plant.PCR reaction systems are：10-50ng/ Ul genomic templates；10 μM of each 0.5 μ l of PC1 and PC2；2.5μl 10×Reaction Buffer；4μl dNTP mix (2.5mM)；0.5 μ l Taq enzymes, add water to 25 μ l.PCR primer detects through 1% agarose gel electrophoresis.There are 36 plants can expand Go out 476bp purpose band, be accredited as positive plant (Fig. 2).

The copy number analysis of the transgenic wheat of embodiment 3

Southern hybridization kits are Roche Holding Ag's product, and hybridization probe uses digoxigenin labeled.Southern hybridizes As a result (Fig. 3) is shown, digestion is carried out to transgenic line OsT5-28 STb gene using tri- kinds of enzymes of BamHI, KpnI and SacI, led to Cross and hybridize with probe, as a result show that OsT5-28 strains contain the target gene of 2 copies, target gene OsPHR2 probe sequences It is identical with PCR identifications sequence.Labeling method and crossover process are as follows：

1) extracting genome DNA uses CTAB methods；

2) DNA enzymatic is cut：100 μ l systems add DNA20 μ g water-bath digestion 12-16 hours；

3) digestion system electrophoresis:0.7% Ago-Gel 1*TAE electrophoresis liquid 35V/12 hours；

4) During migration is using conventional transferring film or the downward transferring film method of vacuum upwards；

5) crossover process：

A：The hybridization buffer of appropriate volume is preheating to hybridization temperature (37-42 DEG C) in advance.

Film reverse side is adjacent to hybrid pipe, adds 5-10ml hybridization solutions, gently vibration 30-60 minutes are carried out in advance in hybrid heater Hybridization；

B：(concentration is about 25ng/mL to the DNA probe of denaturation digoxigenin labeled in digoxin hybridization solution, uses advance Row probe labelling efficiency detects) probe is put into boiling water bath boil 5 minutes after be quickly put into mixture of ice and water and cool down；

C：The digoxin labelled probe of denaturation is added to digoxin hybridization buffer (the film addition per 100cm2 of preheating 5ml hybridization buffers) in, be sufficiently mixed but to avoid producing foam (bubble easily produces background)；

D：Prehybridization solution is poured out, adds the mixture of probe hybridization solution into hybrid pipe immediately.Gentle oscillation incubation 10-16 Hour.

6) membrane process is washed

A：Forbid to wash

A. washed twice with 20ml 2 × SSC 0.5%SDS continuous oscillations, 5 minutes every time；

B.65-68 DEG C, two are washed with enough 0.5 × SSC 0.5%SDS (being preheating to wash temperature in advance) continuous oscillation It is secondary, 15 minutes every time.

B：Immune detection

A.15-25 DEG C, with lavation buffer solution flushing membrane 1-5 minutes；

B. it is incubated 30 minutes in 100ml blockades liquid；

C. it is incubated 30 minutes in 20ml antibody-solutions；

D. washed twice with 100ml lavation buffer solutions, 15 minutes every time；

E. 2-5 minutes are balanced in 20ml detects buffer solution；

F. by film carry DNA one side upward, be placed on the preservative film of tiling, to film on add 1ml at any time i.e. use CSPD.Immediately with preservative film cover to film, uniformly paving substrate, and there can not be bubble on film.5 points are incubated in 15-25 DEG C Clock；

G. remove unnecessary liquid, the edge of folded clip all sealed, pay attention in process-exposed film done can cause it is black The generation of background；

H. it is incubated 10 minutes under the conditions of film being placed on into 37 DEG C, to strengthen luminescence-producing reaction；

I. under the conditions of 15-25 DEG C, exposed about 5-20 minutes using imager, or divided with X-ray exposure 15-25 Clock, notice that chemiluminescence continues at least 48 hours.Signal constantly strengthens in several hours after detection reaction starts, will Reach a threshold value, signal intensity at this moment can almost continue to ensuing 24-48 hours.

7) probe mark and determination (the DIG-High Prime DNA Labeling and of labeling effciency efficiency Detection Starter Kit II, Roche companies, article No. 11585614910) (probe marked region is shown in gene order).

A. 1 μ l points are taken out on nylon membrane from the probe of mark and the 2-9 pipes of mark comparison DNA respectively；

B. by UV-crosslinked method by cDNA chip on film；

C. oscillation incubation 2 minutes in 15-25 DEG C are equipped with the hybrid pipe of 20ml maleate buffers film transfer to one；

D. it is incubated 30 minutes in 10ml blockades liquid；

E. it is incubated 30 minutes in 10ml antibody-solutions；

F. washed twice with 10ml lavation buffer solutions, 15 minutes every time；

G. 2-5 minutes are balanced in 10ml detects buffer solution；

H. by film carry DNA one side upward, be placed on the preservative film of tiling, to film on add 1ml at any time i.e. use CSPD.Immediately with preservative film cover to film, uniformly paving substrate, and there can not be bubble on film.5 points are incubated in 15-25 DEG C Clock；

I. remove unnecessary liquid, the edge of folded clip all sealed, pay attention in process-exposed film done can cause it is black The generation of background；

G. it is incubated 10 minutes under the conditions of film being placed on into 37 DEG C, to strengthen luminescence-producing reaction；

K. under the conditions of 15-25 DEG C, exposed about 5-20 minutes using imager, or divided with X-ray exposure 15-25 Clock, notice that chemiluminescence continues at least 48 hours.Signal constantly strengthens in several hours after detection reaction starts, will Reach a threshold value, signal intensity at this moment can almost continue to ensuing 24-48 hours.

The transcription situation analysis of the transgenic wheat of embodiment 4

CDNA templates are diluted 10 times, divided by extraction control and the total serum IgE of Transgenic plant of wheat, reverse transcription into cDNA Not carry out OsPHR2 genes and actin genes RT-PCR amplifications.Template consumption is adjusted with actin genes, when amplified production When brightness is basically identical, it may be determined that template concentrations are consistent, for OsPHR2 gene magnifications.

Amplification is not as shown in figure 4, adjoining tree amplifies purpose band, and Transgenic plant of wheat amplifies 476bp target stripe, show that OsPHR2 genes have obtained correct transcription in Wheat volatiles.

The expression analysis of the transgenic wheat of embodiment 5

1) sample treatment

A：Protein quantification is carried out with BCA kits to protein sample；

B：According to SDS-PAGE applied sample amount needs, protein concentration is adjusted with water and 5 × loading buffer, 100 DEG C are boiled Boiling denaturation 5min.

2)SDS-PAGE

A：According to the molecular weight of destination protein, 8% separation glue gel is prepared, concentration gum concentration is 5%；

B：Protein sample applied sample amount to be detected：The μ g of loading 30；

C：Deposition condition：Concentrate glue constant pressure 80V, about 20min；Separation gel constant pressure 100V, electrophoresis to bromophenol blue to gel bottom Portion.

3) transferring film

A：Transferring film method：Semidry method；

B：Transferring film condition：During using semidry method, according to electric current 1.2mA/cm2 membrane areas, constant current transferring film；0.45 μm of aperture NC Film, transferring film time 1h；

C：Ponceaux staining reagent (Cat# is used after transferring film：CW0057) film is dyed, observes transferring film effect.

4) Western blot testing goals albumen

A：Closing：Film is immersed in 5% skimmed milk power (TBST dissolving, pH7.5) to (confining liquid must cover room temperature jog 1h Cover whole film, and film can shake wherein be advisable)；

B：Primary antibody is incubated：Primary antibody is diluted with confining liquid, is incubated at room temperature 30min, puts 4 DEG C overnight；

C:Wash film：TBST washes film 3 times, each 10min；

D：Secondary antibody is incubated：Secondary antibody, goat-anti rabbit-HRP1 are diluted with Western blot confining liquids II:5000, room temperature jog 40min；

E：Wash film：TBST washes film 3 times, each 3min；

F：ECL reactions, exposure：The film of one 8cm × 6cm size is anti-with 1ml ECL (the μ lB liquid of 500 μ lA liquid+500) 3min, exposure 1min (time for exposure adjusts with different luminous intensities) are answered, develop 2min, is fixed；

G：Wash film：TBST washes film 3 times；

H：Closing：Film is immersed in room temperature jog 30min in Western blot confining liquids；

I：It is incubated internal reference：Add Beta-actin mouse monoclonal antibodies, with Western blot confining liquids dilute antibody, 1：3000 is dilute Release, 12ml systems, 4 DEG C are put in overnight after being incubated at room temperature 30min；

J：Wash film：TBST washes film 5 times, each 3min；

K：Secondary antibody is incubated：Secondary antibody, sheep anti-Mouse-HRP1 are diluted with Western blot confining liquids:5000, room temperature jog 40min；

L：Wash film：TBST washes film 5 times, each 3min；

M：ECL reactions, exposure：The film of one 8cm × 6cm size is with 1ml ECL (the μ l B liquid of 500 μ l A liquid+500) 3min is reacted, exposure 10s (time for exposure adjusts with different luminous intensities), is developed, is fixed.

Transgenic wheat boot leaf Western hybridization analysis (Fig. 5) shows that positive band occur in OsT5-28 strains, Rather than transgenic wheat does not occur positive band, show that target gene OsPHR2 is obtaining stabilization just in wheat transgenic plant Really expression.

The transgenic wheat P acquisition utilization level of embodiment 6 is analyzed

1) Root Characters are analyzed

By solution culture, full phosphorus (phosphorus concentration 0.2mmol/L), low-phosphorous (0.1mmol/L) and scarce phosphorus (0mmol/ are set L) three processing, 3 repetitions is each handled, are sampled after being cultivated 20 days in nutrient solution, transgenosis is determined using root scanner Wheat and the root long (L) of control.It can be seen from figures 6 and 7 that the most long root long of transgenic wheat is in low-phosphorous and scarce phosphorus condition Under obviously higher than control, under the conditions of full phosphorus with contrast difference unobvious.The root fresh weight of transgenic wheat is in low-phosphorous and scarce phosphorus bar Slightly above compareed under part.Show that OsPHR2 genes can improve the root feature of wheat, turn OsPHR2 DNA triticums so as to improve P acquisition utilization ratio.

2) agronomic characteristics are investigated and analysed

Zheng wheat 0856 field growing way no significant difference, shows as compared with its transgenosis system：Semi-winterness medium variety, children Seedling is partly crawled, and seedling leaf is slightly narrow, and leaf color is light green, and growing way is strengthened, and winter winter resistance is good；Adult plant plant type is elastic moderate, and boot leaf is inclined It is big upper to lift, save short under fringe, ventilation and penetrating light is good between plant；Average plant height 75.4cm, stalk is sturdy, and elasticity is preferably, more resistant to lodging； The big fringe of spindle-type, long awns, fringe layer are neat；Seed cutin, uniform in size, black embryo is few, and plumpness is good, seed outward appearance commodity compared with It is good；Yield trials result shows (table 1)：Zheng wheat 0856 turn of OsPHR2 DNA triticum new lines OsT5-28, OsT5-167, OsT5- 306, compared with wild type control increase yield significantly, analyze the discovery of its yield component, the raising of grain number per spike and mass of 1000 kernel is its volume increase Principal element.

Table 1：Transgenic wheat new lines are with compareing yield trials result

Claims (6)

1. applications of the low phosphorus stress on rice transcription factor OsPHR2 in wheat P acquisition utilization ratio is improved, its feature exist In：By the way that low phosphorus stress on rice transcription factor OsPHR2 is imported into Common Wheat Varieties Zheng wheat 0856 using particle gun mediated method In, obtain stable heredity and the Transgenic plant of wheat expressed.

2. application according to claim 1, it is characterised in that comprise the following steps：

(1) structure of OsPHR2 genes linear expression vector；

(2) by the linear carrier transformed wheat callus containing target gene；

(3) the PCR identifications of resistance regeneration plant.

3. application according to claim 2, it is characterised in that：Step (1) is to contain OsPHR2 bases using HindIII digestions The ring-type expression vector pWMB003-OsPHR2 of cause, obtains the linear carrier containing target gene, piece segment length 3kb, be containing The linear minimum expression cassette of corn Ubiquitin promoters, OsPHR2 genes and Nos terminators.

4. application according to claim 2, it is characterised in that：Step (2) be using the rataria of Zheng wheat 0856 as acceptor material, After MS inducing culture light cultures 4-5d, hypertonic culture medium infiltration processing 4-6h is placed in, target gene linear carrier will be contained With containing bar gene linear carriers, using particle gun mediated method cotransformation WHEAT CALLUS, continue to locate on hypertonic culture medium 16h is managed, goes to calli induction media, culture goes to careless fourth phosphine resistance screening regeneration culture medium, the generation of step sizing 2, sieve after 2 weeks The resistance seedling of choosing goes to root media, takes root, is transplanted after strong sprout, obtains resistance regeneration plant.

5. application according to claim 4, it is characterised in that：

Described MS inducing cultures are：The sucrose of MS+2mg/L 2,4-D+0.4% agar+3%, pH6.2；

Described hypertonic culture medium is：MS+2mg/L 2,4-D+0.7% agar+sucrose of mannitol+3%, pH5.8；

Described careless fourth phosphine resistance screening regeneration culture medium is：1/2MS+0.5mg/L NAA+2.0mg/L KT+0.4% agar+ 3% sucrose+4%PPT, pH6.2；

Described root media is：1/2MS+0.2mg/L the sucrose of NAA+0.4% agar+3%, pH6.5.

6. application according to claim 2, it is characterised in that：Step (3) is to extract the genomic DNA of regeneration plant, root A pair of specific primer PHR2P1 are devised according to OsPHR2 gene orders：5'-AATATGGAGGCTTTGAACC-3' and PHR2P2： 5'-TAACACACCCTTGGGAGTA-3', amplification program are：95 DEG C 2 minutes；95 DEG C 20 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 38 Individual circulation；72 DEG C 10 minutes, purpose fragment length is 476bp；PCR reaction systems are：10-50ng/ μ l genomic templates；10μM PHR2P1 and each 0.5 μ l of PHR2P2；2.5μl 10×Reaction Buffer；The 2.5mM μ l of dNTP mix 4；0.5μl Taq enzyme, add water to 25 μ l.